Molecular epidemiology analysis of fowl adenovirus detected from apparently healthy birds in eastern China

Background Fowl adenovirus is of major concern to the poultry industry worldwidely. In order to monitor the prevalent status of Fowl adenovirus in China, a total of 1920 clinical samples from apparently healthy birds in the 25 sites of poultry flocks, Slaughterhouse and living bird markets from 8 provinces in eastern China were collected and detected by PCR, sequencing, and phylogenetic analysis. Results The epidemiological survey showed that Fowl adenoviruses were detected in living bird markets, and circulating in a variety of fowl species, including chickens, ducks, goose and pigeons. Among the 1920 clinical samples, 166 samples (8.65%) were positive in the fowl adenovirus PCR detection. In this study, totally all the 12 serotypes (serotypes of 1, 2, 3, 4, 5, 6, 7, 8A, 8B, 9, 10 and 11) fowl adenoviruses were detected, the most prevalent serotype was serotype 1. Phylogenetic analysis indicated that 166 FAdVs of 12 serotypes were divided into 5 fowl adenovirus species (Fowl aviadenovirus A, B, C, D, E). Conclusions In the epidemiological survey, 8.65% of the clinical samples from apparently healthy birds were positive in the fowl adenovirus PCR detection. Totally all the 12 serotypes fowl adenoviruses were detected in a variety of fowl species, which provided abundant resources for the research of fowl adenoviruses in China. The newly prevalent FAdV serotypes provides valuable information for the development of an effective control strategy for FAdV infections in fowls.

In this study, a total of 1920 clinical samples were collected from apparently healthy birds in 25 poultry flocks, Slaughterhouse and living bird markets (LBMs) from 8 provinces in eastern China. This is a comprehensive survey from apparently healthy fowls. FAdVs were detected and serotyped to investigate the prevalence of FAdV and analyze the genetic epidemiology.

Epidemiological analysis
Among the 1920 clinical samples, 166 samples (8.65%) were positive in the FAdV PCR detection. Spatial analysis showed that, except Hebei province, FAdVs were detected in all the other 7 provinces, including Hubei, Jiangxi, Shanghai, Guangxi, Jiangsu, Guangdong, Anhui (Fig. 1). The FAdV infection rate in Huhei province is the highest (23.75%). The details were showed in Table 1. All the FAdVs were detected from LBMs. All the samples collected from large-scale farms were negative in PCR detection. In the samples collected from 5 kinds of fowl, FAdV was detected in samples of chicken (13.27%), goose (2.50%), duck (0.48%) and pigeon (0.43%), while negative in the samples of partridge.  Table 1 The number and distribution of the 12 FAdV serotypes detected in the 8 provinces  In 166 cases, 18.07% (30/166) of the isolates were related to FAdV-A (including serotype 1), and 3.61% (6/166) of the isolates were identified as FAdV-B (including serotype 5), and 21.69% (36/166) of the isolates were identified as FAdV-C (including serotypes 4 and 10), and 38.55% (64/166) of the isolates were identified as FAdV-D (including serotypes 2, 3, 9, and 11), and 18.07% (30/166) of the isolates were identified as FAdV-E (including serotypes 6, 7, 8A and 8B). The prevalent rate of circulating species was showed in Table 3.

Phylogenetic analysis of FAdVs
The results of the phylogenetic analysis of FAdVs detected in this study and reference strains are shown in Fig. 2. In the analysis, the FAdV strains clustered into five major groups. Cluster 1 is corresponding to Fowl aviadenovirus D (FAdV-D), including serotypes 2, 3, and 11 FAdVs. Cluster 2 is corresponding to Fowl aviadenovirus Table 2 The number and distribution of the 12 FAdV serotypes detected in 5 kinds of poultry  Table 3 The prevalence rate of circulating species and serotypes in this study

Discussion
FAdVs are commonly present in fowl farms worldwide [17]. As reported, more and more FAdVs have been isolated from dead or sick animals in recent years [2,6,[18][19][20]. FAdV infections are associated with a range of avian infectious diseases, such as IBH and HHS.
In China, since 2015, sporadic outbreaks of HHS occurred with suddenly high mortality rates in layers in most areas in China [7]. It was previously reported that the FAdV infection was caused by a variety of different FAdV species in China, at least two or three species of FAdVs (species C, D or E) were detected [7,11]. In our survey, the present epidemiology surveillance showed more abundant diversity and wider distribution than the previous reported studies in China [7,12,14], the surveillance showed that all the five species (species A, B, C, D and E) FAdVs were detected in the FAdV survey in 7 provinces (Jiangxi, Shanghai, Guangxi, Jiangsu, Fig. 2 Phylogenetic analysis of FAdVs detected in this study and reference strains. Phylogenetic relationships were calculated using the model with the Maximum likelihood (ML). Gaps were handled by pairwise deletion and bootstrap values were calculated from 1, 000 replicates. FAdV strains in this study were marked with "▲" Guangdong, Anhui, Hebei) of China. Significantly, all the detected samples in this survey were collected from apparently healthy birds, no diseased or dead fowls were sampled. This is a comprehensive survey from apparently healthy fowls. It has great significance for the prevention and control of circulation of fowl adenovirus in China.
Among all the circulating FAdV species, FAdV-D showed the highest percentage of 38.55% (including serotypes 2, 3, 9, and 11) in this study, which is different with the previous study [4,11]. In this study, FAdV-D strains mainly circulating in Southern provinces of China, which including Anhui, Guangdong, Guangxi, Hubei, Jiangsu, Jiangxi, and Shanghai. Meanwhile, the prevalence rate of FAdV-A, FAdV-C and FAdV-E is maintained between 18.07% and 21.69% in southern China. Although the prevalent rate of species FAdV-A, FAdV-C and FAdV-E was lower than the dominant species FAdV-D, the strict biosecurity measures may be necessary to the prevention and control of FAdVs.
The results showed that all the FAdV-D strains and the most prevalent serotype 1 strains were detected from chickens in LPMs, no positive samples were detected from slaughterhouse or large-scale farms. This indicated that chicken may be an important risk, which indicated that chickens in the LPMs of southern China may play an important role in the transmission and circulation of FAdVs. According to the relevant reports around the world, duck adenovirus have been reported since 2014 [21][22][23][24][25][26], and infection of FAdV-4 in geese and pigeon adenovirus 1 have been reported in the previous study [27][28][29]. Meanwhile, FAdVs were also detected from ducks, geese and pigeons in this study. The role of waterfowl and birds (e.g. pigeon) in the spread of FAdVs should be paid more attention in the further study. All the cloacal/throat double swabs in this study were collected from clinical healthy birds according to a random sampling method. The results showed that 8.65% collected samples were positive in the FAdV detection. As reported, it is believed that the mechanism of FAdV infection is very complex in chicken flocks [7]. The pathogenicity, such as FAdV-D and FAdV-E, was not evaluated and, thus, further investigation are warranted.

Conclusion
Taken together, in the FAdV epidemiological survey, 8.65% of the clinical samples from apparently healthy birds were positive in the fowl adenovirus PCR detection. Totally all the 12 serotypes fowl adenoviruses were detected in a variety of fowl species, which provided abundant resources for the research of fowl adenoviruses in China. The present study provides new information about the epidemiology and characteristics of fowl adenoviruses associated with chickens, ducks and pigeons in China, which will provide a basis for further understanding of the disease, and would aid in the prevention and control of FAdVs. Surveillance for fowl adenovirus must be continued worldwide.

Sample collection and Nucleic acid extraction
In 2019, totally 25 sites of large-scale farms, slaughterhouse and LBMs of the 8 provinces (Hubei, Jiangxi, Shanghai, Guangxi, Jiangsu, Guangdong, Anhui, Hebei) in China were selected at random. Cloacal/throat double swabs were collected from more than 60 clinical healthy birds at each farm according to a random sampling method. Totally, 1920 swab samples were collected, the sample details were listed in Table 4. The samples were stored at 4 °C by a preservation buffer of 1.2 mL

Screening of FAdV in clinical samples
In order to screen and analyze FAdV, conventional PCR using degenerate primers specific for the L1 loop of the FAdV hexon genes was applied according to the published report [30]. The primers were designed by alignment comparison of the conserved sequences of FAdV1, FAdV8 and FAdV9, and were able to amplify the 12 serotypes of FAdVs [30]. As reported in the previous studies [4,14,16], it is shown that the amplification of the Hexon gene by molecular biology method can also be used to identify the serotype of FAdVs. We identified the serotype of FAdVs by using molecular biology methods in this study.

Sequencing
PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), cloned into the pMD18-T vector (Takara, Dalian, China), and then sequenced using synthetic oligonucleotides (Sangon Biotech, Shanghai, China). The sequences were submitted to the GenBank database, National Center for Biotechnology Information.

Phylogenetic analysis
All the hexon sequences derived from the detected FAdV were aligned with 22 selected hexon genes of the 12 serotypes FAdV reference strains in the ICTV classification system and some other selected field strains, following the published reports [30]. Hexon gene sequences of the representative strains were downloaded from NCBI, GenBank accession numbers of these sequences were listed in Table 5. The phylogenetic tree based upon the results of multiple sequence alignment was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 [31,32] applying the model with the maximum likelihood (ML) method, The robustness of the phylogenetic constructions was evaluated by bootstrapping with 1,000 replicates [31]. Serotypes were identified based on the phylogenetic analysis and pairwise identities as described in previous studies [30].